The physical and chemical properties of socium channels in nerve and muscle membranes will be studied at the molecular level. The highly selective neurotoxins, tetrodotoxin and saxitoxin, will be used to probe that portion of the sodium channel to which they bind. Saxitoxin (STX) in particular will be used because it can be labelled to very high specific radioactivity (hr.STX). Since the purity of the toxin molecules actually bearing the isotopic label can be checked by NMR spectroscopy the toxin will become a standard for the comparison of all other toxins. The importance for binding of specific chemical groups on STX will be tested by isotopic-exchange studies, by the pH-dependence of the action of STX on nerves, and by the action of chemically modified STX derivatives. Groups on the sodium channel which are important for toxin binding will be identified by competition/protection experiments using amino acid-selective reagents and the selective neurotoxins. Similar experiments using permeant and impermeant cations to protect sodium currents from the action of amino acid-selective reagents will be used to construct a "map" of identifiable groups at the mouth of the sodium channel. Both saxitoxin and tetrodotoxin will be used to study the changes in sodium channels of muscle following denervation. The spatial distribution of sodium channels on normal and denervated muscle will be studied by using autoradiography of hr.STX bound to these cells. Concomitant with these studies will be experiments to covalently link the STX to the sodium channel. Binding of STX to single cells and voltage-clamped axons will be used for the direct calculation of the conductance of single, open sodium channels. Extended aspects of this research proposal include the development of an antidote to tetrodotoxin and saxitoxin poisoning and the attempted isolation and purification of the sodium channel by affinity chromatography.